Multinuclear complexes for X-ray imaging

ABSTRACT

The invention relates to the use as contrast enhancing agents in medical imaging, especially X-ray imaging, of multinuclear complexes, i.e. complexes, such as those of W2O2 (u2O)2, in which the complexed entity comprises at least two contrast enhancing atoms.

This application is a 371 of PCT/EP91/00587 filed on Mar. 27, 1991.

The present invention relates to the use in diagnostic imaging, inparticular X-ray, ultrasound and scintigraphy of contrast agentscomprising complexes of multinuclear moieties, and to contrast mediacontaining such complexes.

All diagnostic imaging is based on the achievement of different signallevels from different structures within the body. Thus in X-ray imagingfor example, for a given body structure to be visible in the image, theX-ray attenuation by that structure must differ from that of thesurrounding tissues. The difference in signal between the body structureand its surroundings is frequently termed contrast and much effort hasbeen devoted to means of enhancing contrast in diagnostic imaging sincethe greater the contrast between a body structure and its surroundingsthe higher the quality of the images and the greater their value to thephysician performing the diagnosis. Moreover, the greater the contrastthe smaller the body structures that may be visualized in the imagingprocedure, i.e. increased contrast can lead to increased spatialresolution.

The diagnostic quality of images is strongly dependent on the inherentnoise level in the imaging procedure--and the ratio of the contrastlevel to the noise level can thus be seen to represent an effectivediagnostic quality factor for diagnostic images.

Achieving improvement in such a diagnostic quality factor has long beenand still remains an important goal. In techniques such as X-ray andultrasound, one approach to improving the diagnostic quality factor hasbeen to introduce contrast enhancing materials, contrast agents, intothe body region being imaged.

Thus in X-ray for example early examples of contrast agents wereinsoluble inorganic barium salts which enhanced X-ray attenuation in thebody zones into which they distributed. More recently the field of X-raycontrast agents has been dominated by soluble iodine containingcompounds such as those marketed by Nycomed AS under the trade namesOmnipaque and Amipaque.

Much recent work on X-ray contrast agents has concentrated onaminopolycarboxylic acid (APCA) chelates of heavy metal ions and,recognising that effective imaging of many body sites requireslocalization at the body sites in question of relatively highconcentrations of the metal ions, there have been suggestions thatpolychelants, that is substances possessing more than one separatechelant moiety, might be used to achieve this.

However we have now found that contrast enhancement may be achievedparticularly effectively by the use of multinuclear complexes, that iscomplexes wherein the complexed moiety itself comprises two or morecontrast enhancing atoms or for X-ray or ultrasound two or more heavyatoms.

For the sake of clarity, the word "atom" is used to refer to ionic andcovalently bonded forms and not simply to isolated uncharged atoms.Moreover it will be understood that the complexed moiety, while it ispolynuclear, is not so large as to be considered to be a particleitself. Thus it will generally have maximum dimensions of 80 Å or less,especially 40 Å or less.

Thus viewed from one aspect the invention provides a method ofgenerating an image of a human or non-human animal, preferablymammalian, body which method comprises administering to said body aphysiologically tolerable contrast enhancing amount of a multinuclearcomplex and generating an image of at least part of said body, e.g. byX-ray, ultrasound, or scintigraphy.

Viewed from a further aspect the invention also provides a multinuclearcomplex, especially a tungsten and/or molyblenum complex, for use as adiagnostic image contrast enhancing agent.

Viewed from a still further aspect the invention also provides adiagnostic imaging contrast medium comprising a multinuclear complextogether with at least one sterile pharmaceutical carrier or excipient.

Viewed from another aspect the invention provides the use of amultinuclear complex for the manufacture of a contrast medium for use inimaging of the human or non-human animal body.

Multinuclear complexes have particular potential as contrast agentssince, relative to mononuclear complexes such as the paramagnetic metalion APCA chelates and polychelates conventionally proposed for use asX-ray contrast agents, the increase in the contrast enhancing atomcontent of the molecule is achieved with relatively little increase inthe volume occupied by the contrast agent complexes, that is to say theuse of multinuclear complexes enables a high ratio of contrast enhancingatom to overall complex volume to be achieved. Thus by increasing therelative content of contrast enhancing atoms in this way the totalquantity of the contrast agent necessary in order to achieve the samecontrast effect may be reduced and thus problems associated withcontrast agent solubility or toxicity or with contrast medium viscositymay also be reduced.

The multinuclear complex used according to the invention may be ionicor, more preferably, may carry no net charge; most preferably thecomplex is non-ionic. Moreover it may be water-soluble or, lesspreferably, water-insoluble. Any necessary counterions should of coursemost preferably also be physiologically tolerable.

The range of physiologically acceptable counterions for therapeuticallyactive agents is of course well known to pharmacologists.

Suitable counter cations include for example alkali and alkaline earthmetal ions, e.g. sodium, calcium and magnesium and zinc, ammonium andorganic amine cations, e.g. meglumine, alkylammonium,polyhydroxyalkylammonium, basic protonated amino acids, etc. Suitablecounteranions include for example halide (e.g. chloride, bromide oriodide), sulphate, mesylate, phosphate, etc.

As mentioned above, by multinuclear it is meant that the complexedmoiety should comprise two or more contrast enhancing atoms (preferablyin the form of a molecular ion or ion groups). The multinuclear moietymay thus optionally contain further atoms which may have little or nocontrast enhancing effect but which may for example function as bridgingatoms bonding the contrast enhancing atoms together. Particularlysuitable examples of bridging atoms include those of group VIb, e.g.oxygen, sulphur, selenium and tellurium, and substituted nitogen atoms.The use of selenium and tellurium, e.g. as bridging atoms, is especiallyattractive since the X-ray cross sections of these atoms, especiallytellurium, are greater than those of the lower atomic weight sulphur,oxygen and nitrogen accordingly such atoms will contribute substantiallyto the overall X-ray attenuation by the complex.

Preferably the complexed multinuclear moiety will contain at least 2,for example up to 30, such as 2-15, e.g. 2 to 6, preferably 2 to 5contrast enhancing atoms, particularly preferably 2, 3 or 4. Theappropriate nature, e.g. the element, the isotope or the oxidationstate, of the contrast enhancing atoms is of course dependent on theimaging technique in which the multinuclear complex is intended tofunction as a contrast agent. Thus for X-ray and ultrasound imaging thecontrast enhancing atoms conveniently have atomic members of at least37, preferably at least 50, and for scintigraphy the contrast enhancingatoms will be radioactive isotopes, e.g. radioactive metal ions.

For use as an X-ray contrast agent, it will generally be preferred thatthe multinuclear moiety should contain two or more heavy metal atoms,e.g. lanthanide, transition metal or other metal atoms such as forexample Ce, Hg, Sr, Y, Zr, Tc, Ru, In, Ta, Nb, Dy, Hf, W, Mo, Re, Os,Pb, Ba, Bi, Ga, Sn and Tl, however Mo and W are particularly preferred.The choice of heavy metal used in the multinuclear complexes will bedetermined by a variety of factors including the toxicity of the overallcomplex and the X-ray absorption characteristics of the heavy atom. Inthis regard it should be noted that while the X-ray absorption crosssection for atoms generally increases with increasing atomic nuber, theabsorption cross section is itself dependent on the X-ray wavelength andincreases with increasing photon energy until slightly above a valuetermed the K-edge whereafter attenuation decreases. Thus there arephoton energy ranges for which one element is a better X-ray attenuatorthan a second even though outside these ranges the second element may bethe better attenuator. Consequently the multinuclear complexes accordingto the invention will each have optimum photon energy ranges making themparticularly suitable for operation with X-ray imaging apparatusutilizing X-rays having such photon energy ranges. However, by choosingmultinuclear complexes containing atoms of more than one heavy elementone may create X-ray contrast agents having optimal performance in morethan one photon energy band or over a broader band. The complexes usedaccording to the present invention are thus particularly attractivesince they can be selected so as to match their X-ray attenuationprofiles with the X-ray emission profiles of particular X-raysources--in effect the invention provides "tunable" X-ray contrastmedia.

Non-chelant complexing agents, such as amines and carboxylic acids, e.g.acetic acid and amino acids, are known and may be used in the formationof the multinuclear complexes of the invention. However since many ofthe contrast enhancing multinuclear entities are extremely toxic it isclearly preferable that the formation constants of the multinuclearcomplexes should be as high as possible and accordingly it isparticularly preferred that the multinuclear moiety should be bound in achelate complex. Suitable chelant moieties will be discussed furtherbelow.

Many multinuclear complexes are known and attention is drawn for exampleto the following publications: Chisholm, Trans. Met. Chem. 3: 321(1978); Lee et al., Ang. Chem. Intl. Ed. Eng. 29: 840-856 (1990); theAbstracts of the 5th International Conference on the Chemistry and Useof Molybdenum, 1985, page 133; Novak et al., J. Inorg. Nucl. Chem. 36:1061-1065 (1974); Burgi et al., Inorg. Chem. 20: 3829-3834 (1981);Chaudhuri et al., Z. anorg. allg. Chem. 521: 23-36 (1985); Ikari et al.,Inorg. Chem. 29: 53-56 (1990); Tomohiro et al., J. Chem. Soc. DaltonTrans. 1990, 2459-2463; Henkel et al., J. Chem. Soc. Dalton Trans. 1990,1014-1016; Barbaro et al. JACS 112:7238-7246 (1990); Richens et al.,Inorg. Chem. 28: 1394-1402 (1989); Saito et al., Inorg. Chem. 28:3588-3592 (1989); J. Chem. Soc. Dalton Trans 1990, 1765-1769; Inorg.Chem. 27:3626- 3629 (1988); JACS 108:2757-2758 (1986); and referencescited therein. These multinuclear complexes generally fall into twocategories, those in which the multinuclear moiety is bridged, that isto say where liganded metal atoms are bonded together via other atoms orligands, and those where the moiety is unbridged, i.e. where theliganded metal atoms are bonded together directly. There is also a thirdgeneral category in which the multinuclear moiety is apparently notbonded together, e.g. where two or more separate metal ions arecomplexed by the same chelant moiety.

Thus for example for the use of multinuclear complexes containing twoliganded metal atoms (M) the main alternative structures are ##STR1##where each M which may be the same or different is a metal atom; each Lwhich may be the same or different is a ligand, either a molecule, anion or one liganding moiety of a multidentate ligand; each B which maybe the same or different is a bridging atom or ligand; and each m is aninteger. Several L groups can of course be provided by one chelant andthe metal atoms may be covalently bound to further atoms (generallydesignated by the letter A in the formulae referred to herein) notindicated by L or B and which function neither as ligands nor asbridges.

Where the M-B bonds to the bridging groups B of formula II arecoordinate rather than covalent bonds, the multinuclear complex will beof the third general category referred to above. Examples of suchcomplexes thus include the macrocyclic binuclear chelates such as##STR2## where each R which may be same or different is hydrogen or anorganic group and each M which may be the same or different is a metalatom or ion, e.g. Ni, Pb(II) or Cu(II).

Where the liganded metal atoms are directly bonded, the MM distancestend to be short and the multinuclear complexes are generallydiamagnetic. Several complexes falling into this category are wellknown, e.g. compounds of formulae

    L.sub.5 MML.sub.5                                (Ia)

    L.sub.4 M.tbd.ML.sub.4                                     (Ib)

(where each M which may be the same or different is for example Mo orRe).

While the use of multinuclear complexes wherein the liganded atoms arebonded directly together or are not linked by covalent bonds does fallwithin the scope of the invention, it is particularly preferred that themultinuclear complexes be of the bridged type wherein the liganded metalatoms are covalently linked via bridging atoms. Many such complexes areknown and typical exemplary structures include the bi-, tri-, tetra- andhexa- nuclear structures of formulae II, V, VIII and IX ##STR3## whereeach m which may be the same or different is an integer, each B whichmay be the same or different is a bridging atom and each M which may bethe same or different is a metal, e.g. Mo, W, Re, or Tc, and where othernon-bridging atoms covalently bonded to metals M are omitted for thesake of clarity. These bi, tri, tetra and hexanuclear clusters offormula M₂ B₂, M₃ B₄, M₄ B₄, and M₆ B₈ are well described in theliterature, see for example

J. Chem. Soc. A. 1970, 2421;

JCS Dalton Trans. 1975, 1526-1530;

Inorg. Chem. 16: 2538-2545 (1977);

JACS 99: 4168-4169 (1977);

J. Inorg. Nucl. Chem. 36: 1061-1065 (1974);

Inorg. Chem. 28: 447-451 (1989);

Chem. Letters, 1987, 2327-2330;

J. Chem. Soc. Dalton Trans., 1987, 1163-1167;

Inorg. Chem. 23: 4265-4269 (1984);

Inorg. Chem. 24: 2950-2952 (1985);

C. R. Seances Acad. Sci., Ser. C. 1966, 262, 1524;

JACS 106: 2710-2711 (1984);

J. Chem. Soc. Chem. Comm., 1985, 953;

JACS 107: 5565 (1985);

Inorg. Chem. 27: 3626-3629 (1988);

J. Chem. Soc. Dalton Trans., 1990, 1975-1769;

JACS 108: 2757-2758 (1986);

JACS 106: 789-791 (1984);

JACS 107: 6734-6735 (1985);

Inorg. Chim. Acta 116: L25-L27 (1986);

JACS 105: 3905-3913 (1983);

J. Chem. Soc. Chem. Comm., 1990, 1014-1016;

JACS 112: 7238-7246 (1990);

JACS 110: 1646-1647 (1988);

J. Chem. Soc. Dalton Trans., 1991, 51-59; and

Inorg. Chem. 28: 3588-3592 (1989).

The complexes above may be electrically charged or neutral--foradministration as contrast agents they are however preferably complexedwith ligands/chelating agents which serve to improve water solubilityand to reduce toxicity and to leave unaffected, to only slightlyincrease or, most preferably, to reduce the magnitude of the overallelectronic charge carried by the complex.

In the case of bridged structures of these four formulae, the structuralformulae can conveniently be written M₂ L_(q) (μ₂ B)₂ and M₃ L_(r) (μ₃B)(μ₂ B)₃,M₄ L_(s) (μ₃ B)₄ and M₆ L_(t) (μ₃ B)₈ respectively (μ₃ Bindicating that the B is a bridging atom bonded to 3 metals, and q, r, sand t respectively being integers identifying the total number ofcomplexing moieties). As mentioned above, it is particularly preferredthat the multinuclear complexes be chelate complexes and it isespecially preferred that a single multidentate chelant be used tocoordinate at least two and preferably all of the liganded centres. Amultidentate chelant L coordinating for example three metals would bereferred to in these formulae as (μ₃ L).

Thus the multinuclear complexes used according to the inventionpreferably are compounds of the formula X

    (M.sub.n B.sub.u A.sub.v).sub.x L.sub.w                    (X)

(where M_(n) B_(u) A_(v) is a multinuclear entity; each M which may bethe same or different is a metal atom covalently bonded to at least one,preferably 2-4, atoms; each B which may be the same or different is abridging atom covalently bonded to at least two, preferably 2 or 3,atoms M; each A which may the same or different is a non-bridging atomcovalently bonded to an atom M; each L which may be the same ordifferent is a ligand, preferably a multidentate molecule or molecularion, coordinately bonding to at least one atom M; n and u are positiveintegers of value 2 or greater; x and w are positive integers; and v iszero or a positive integer) or salts, especially physiologicallytolerable salts, thereof.

In formula X above, n, u and v are preferably 2 to 30, especially 2 to10, particularly 2 to 8; x is preferably 1 to 20, especially 1 to 10,and particularly 1. The value of w depends on the size and identity ofthe ligand--nonetheless w is preferably 1 or 2, especially 1.

Particularly preferred multinuclear complexes for use according to theinvention include the APCA chelate complexes of mixed or non-mixed bi,tri, tetra and hexa nuclear oxides, sulphides, selenides and telluridesof molybdenum and/or tungsten, e.g. APCA chelates of multinuclearentities of formula ##STR4## where each M is independently W or Mo andeach Z is independently O, S, Se or Te, e.g. W₂ S₂ (μ₂ S)₂, W₂ O₂ (μ₂O)(μ₂ S), W₂ O₂ (μ₂ S)₂, MoWO₂ (μ₂ O)₂, Mo₂ O₂ (μ₂ O)₂, Mo₂ O₂ (μ₂ S)₂,W₄ (μ₃ S)₄, W₃ (μ₃ S)(μ₂ S)₃, W₃ (μ₃ Se)(μ₂ Se)₃, W₃ (μ₃ Te)(μ₂ Te)₃, W₄(μ₃ Se)₄, W₄ (μ₃ Te)₄, Mo₃ (μ₃ Se)(μ₂ Se)₃, Mo₄ (μ₃ Se)₄, Mo₂ O₂ (μ₂Se)₂, Mo₃ (μ₃ O)(μ₃ O)₃, W₂ O₂ (μ₂ O)₂.

Many of these multinuclear clusters are known from the literature citedabove--the others may be prepared using methods analogous to thosedescribed in the literature.

Particularly conveniently, such multinuclear entities are presented astheir chelate complexes containing EDTA or other APCA's. Such chelatecomplexes are remarkably stable with regard to release of the heavymetal ions and thus W₂ O₂ (μ₂ O)₂ (μ₂ EDTA), for example, has been foundto have a stability constant in aqueous solution of about 29.1 (seeNovak et al., J. Inorg. Nucl. Chem. 36: 1061-1065 (1974)).

The structure of the W₂ O₄ EDTA²⁻, or more preferably [W(V)₂ O₂ (μ₂ O)₂(μ₂ EDTA)]²⁻, multinuclear complex has been suggested by Novak (supra)and others to have the structure ##STR5##

Besides EDTA, other chelants are suitable for the preparation of themultinuclear chelate complexes used according to the invention.

It is particularly preferred that the electrical charge carried by thecomplexing moieties should substantially if not completely balance thatcarried by the complexed entity; for APCA chelants this may easily beachieved for example by omission, replacement or deactivation (e.g. byester or amide formation) of one or more of the carboxyl moieties.

Many suitable chelants are widely known or have been described in theliterature, especially literature relating to heavy metal detoxificationagents bifunctional chelants and chelate-based contrast agents, e.g.those described in WO-A-89/00557 (Berg) and the documents mentionedtherein and in the search report appended thereto, U.S. Pat. No.4,647,447 (Gries), U.S. Pat. No. 4,826,673 (Dean), EP-A-230893 (Felder),EP-A-217577 (Frincke), U.S. Pat. No. 4,652,519 (Warshawsky), U.S. Pat.No. 4,687,659 (Quay), and numerous other recent patent publications ofNycomed AS, Salutar Inc, Schering AG, Squibb, Bracco, Mallinckrodt, Dowand Guerbet.

The chelants useful for complexing the multinuclear moeity can beselected from a wide range of structures. Many of the most usefulchelants are of general formula XIII

    Z'(X(CHR.sub.1).sub.a).sub.b XZ'                           (XIII)

(where a is an integer of from 2 to 12, preferably 2 to 10, e.g. 2, 3,or 4; b is an integer of from 1 to 8, preferably 2, 3 or 4;

each R, independently is hydrogen, a hydrophilic or linking group (e.g.a hydroxyalkyl group) or two groups R₁, or one R₁ and one group Z',together represent a saturated or unsaturated heterocyclic orcarbocyclic ring, preferably with 5-7 ring atoms;

each X independently is O, S, NZ' or PZ', each Z' indpendently ishydrogen, hydroxyalkyl, mercaptoalkyl, carboxyalkyl (or an amide orester derivative thereof e.g. --CH₂ CONHCH₃) or optionally hydroxy ormercapto substituted acyl, or is a side chain ((CHR₁)_(a) X*)_(c) Z*(where c is 1 to 4 and X* and Z* are as form a briding group ((CHR₁)_(a)X*)_(c) (CHR₁)_(a)) or are salts defined for X* and Z* but do notrepresent any group containing a X* or Z* group) or two groups Z'together thereof.

While polyamines, especially linear or cyclic polyamines, such asethylenediamine,1,4,7-triazacyclononane and cyclen, can be used aschelants, in general APCAs are preferred, particularly DTPA, EDTA andderivatives thereof and other cyclic and non-cyclic APCAs as defined inWO-A-89/00557 and APCAs of formula XIV ##STR6## where each R₁ isindependently hydrogen or an optionally hydroxylated and/or alkoxylatedalkyl group or an organic side chain adapted for the attachment of orattached to a macromolecule;

d and e each is an integer having a value of 1, 2 or 3;

each X is independently a group COOH or a derivative thereof;

each Y is independently a group X, SR₁, OR₁ or N(R₃)₂ ;

E is a group (CHR₂)_(f) (X"(CHR₂)_(f))_(g) where f is an integer of from2 to 5, preferably 2 or 3,

g is zero, 1 or 2, preferably zero or 1, each f preferably being 2 wheng is non-zero,

X" is O, S or N(CHR₁)_(d) Y, preferably O or S,

each R₂ is independently R₁ or, when the carbon to which it is attachedis not bonded to a nitrogen, hydroxyl, or two R₂ groups, especiallywhere f is 2, may together with the intervening carbons form acycloalkyl group optionally substituted by hydroxyl or R₁ groups, andeach R₃ is independently a group R₁ or N(R₃)₂ represents a preferablysaturated heterocyclic group preferably having 5 or 6 ring members,optionally containing as a further heteroatom a nitrogen or oxygen andoptionally substituted by R₁ groups.

In the chelants of formula XIII or XIV, any alkyl moiety preferably hasa carbon atom content of up to 8, any cycloalkyl group preferably is aC₃₋₈, especially C₅₋₇, ring and any carboxyl derivative is preferably aCON(R₃)₂ or CON(OH)R₁ group.

Examples of suitable chelants include compounds of formulae:

    __________________________________________________________________________    (HOOCCH.sub.2).sub.2 NCH.sub.2 CH.sub.2 N(CH.sub.2 COOH).sub.2                                                          (i)                                 (HSCH.sub.2 CH.sub.2).sub.2 NCH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2            SH).sub.2                                 (ii)                                H.sub.2 NCH.sub.2 CH.sub.2 N(CH.sub.2 COOH)CH.sub.2 CH.sub.2 N(CH.sub.2       COOH)CH.sub.2 CH.sub.2 NH.sub.2           (iii)                               H.sub.2 NCH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2 SH)CH.sub.2 CH.sub.2           N(CH.sub.2 CH.sub.2 SH)CH.sub.2 CH.sub.2 NH.sub.2                                                                       (iv)                                HOOCCH.sub.2 (NCH.sub.2 CH.sub.2).sub.3 NCH.sub.2 COOH                                                                  (v)                                 HSCH.sub.2 CH.sub.2 (NCH.sub.2 CH.sub.2).sub.4 SH                                                                       (vi)                                 ##STR7##                                 (vii)                               (where y = 6,7,8,9 or 10                                                      and z = 0 or 1)                                                               (HOOCCH.sub.2).sub.2 NH                   (viii)                              (HSCH.sub.2 CH.sub.2).sub.2 NH            (ix)                                (HOOCCH.sub.2).sub.2 NCH.sub.2 CH.sub.2 N(CH.sub.2 COOH)CH.sub.2 CH.sub.2     N(CH.sub.2 COOH)CH.sub.2 CH.sub.2 N(CH.sub.2 COOH).sub.2                                                                (x)                                 (HSCH.sub.2 CH.sub.2).sub.2 NCH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2            SH)CH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2 SH)CH.sub.2 CH.sub.2 N(CH.sub.2      CH.sub.2 SH).sub.2                        (xi)                                (HOOCCH.sub.2).sub.2 N(CH.sub.2 CH.sub.2 NH).sub.2 CH.sub.2 CH.sub.2          N(CH.sub.2 COOH).sub.2                    (xii)                               (HSCH.sub.2 CH.sub.2).sub.2 N(CH.sub.2 CH.sub.2 NH).sub.2 CH.sub.2            CH.sub.2 N(CH.sub.2 CH.sub.2 SH).sub.2    (xiii)                              pyridine-2,6-dicarboxylic acid            (xiv)                               2,6-bis-merceptomethyl-pyridine           (xv)                                 ##STR8##                                                                     tetra-N-alkyl-ethylenediamine             (xix)                               penta-N-alkyl-diethylenetriamine          (xx)                                __________________________________________________________________________

and the phosphorus analogues of these nitrogen-donor based ligands.

For M₄ B₄ multinuclear complexes, e.g. W₄ (μ₃ B)₄ ⁴⁺ (where B═S, Se, Te,O, N--R³¹ or P--R³¹ (where R³¹ is an appropriate substituent, e.g.hydrogen, aryl (e.g. phenyl), alkyl etc.), chelants (i) to (vii) (wherez=1) are of particular interest; for M₃ B₄ complexes, e.g. W₃ (μ₃ B')(μ₂B")₃ ⁴⁺ (where B' and B" are S, Se, Te, O, NR³¹ or PR³¹ B' preferablybeing S) chelants (vii) (where z=o) and (viii) to (xv) are of particularinterest; and for M₆ B₈ complexes, e.g. W₆ (μ₃ S)₈, chelants such as(xvi) to (xx) are of particular interest. For M₂ B₂ complexes, e.f. W₂O₂ (μ₂ O)₂ ²⁺ chelants such as NTA, IDA, EDTA, HEDTA, DTPA, DTPA-BMA,HEDDA, TTDA, EDTA-BMA, TBEDDA, MEEDDA, TTHA, EDDA, EHPG, PDTA, CHDTA,HPDTA and triazacyclononane monoacetic acid, especially PDTA and EDTA,are of particular interest.

For M₄ B₄ and M₃ B₄ multinuclear complexes, the use of macrocyclicchelants, e.g. those of formula (vii) is particularly preferred as ameans by which to enhance solution stability.

Particularly preferred chelants include cyclen, EDTA, DTPA, DOTA, DO3A,HP-DO3A, the 6-oxa and 6-thia analogues of DTPA and amides thereof, e.g.DTPA-BMA and DTPA-BMO(6-carboxymethyl-3,9-bis(morpholinocarbonylmethyl)-3,6,9-triazaundecanedioicacid--the Gd(III) chelate whereof is sometimes referred to asgadopenamide).

Where the chelant is to be attached to a macromolecule, this mayconveniently be any tissue, organ or cell targeting macromolecule, forexample a biomolecule such as a protein, an antibody or antibodyfragment, or alternatively it may be a biologically relatively inertmaterial such as a polysaccharide or poly-sugar alcohol, e.g. dextran orstarch. Such macromolecules are discussed extensively in the recentliterature relating to contrast agents.

The chelants of formulae XIII and XIV are already known from theliterature or may be prepared in analogous fashion to the knownchelants. The preparation of chelants of formula XIII and XIV willhowever generally fall into one of two categories: derivatization of apolyamine or amination of polyfunctional compounds. Derivatization canbe performed in one or more stages and the groups introduced may, inintermediate or final stages, be subject to reduction or deprotectionsteps.

Thus for example starting from the linear polyamine

    NH.sub.2 --E'--NH.sub.2                                    (XV)

(where E' is (CHR₂)_(f) [X"'(CHR₂)_(f) ]_(g) and X"' is O, S or NH)derivatization may be effected by the following nonreductive orreductive reaction schemes: ##STR9## where L is a leaving group and R₁', Y' and X' are optionally protected R₁, Y and X groups.

Alternatively a bifunctional reagent of formulae

    L--E--L                                                    (XVI)

or

    LCO.E".CO.L                                                (XVII)

may be aminated with or without a subsequent reduction step according tothe following schemes: ##STR10## where E" is (CHR'₁)_(f-h) [Z'(CHR₁')_(f) ]_(i) [Z'(CHR'₁)_(f-1) ]_(j) (where j is 0 or 1, h+j is 2, i iszero or the positive integer g-l) and L, R'₁, Y' and X' are ashereinbefore defined.

The polyamine starting materials are either available commercially ormay be prepared by routine methods. Thus for example commerciallysuitable polyamines include NH₂ (CH₂)₂₋₅ NH₂, NH₂ (CH₂)₂ O(CH₂)₂ NH₂,NH₂ CH₂ CHOHCH₂ NH₂, NH₂ (CH)₂ S(CH₂)₂ NH₂. Optionally substitutedpolyamines may also be prepared by methods described in or analogous tothose of EP-A-287465 (Schaeffer), WO-A-89/00557 (Berg), Brechbiel et al.Inorg. Chem. 25: 2772 (1986), Yeh et al. Analytical Biochem. 100: 152(1979), Vogtle et al. Liebigs Ann. Chem. (1977) 1344, Kasina et al. J.Med. Chem. 29: 1933 (1986), Bedell et al. Inorg. Chem. 21:874 ( 1982 ),etc.

Derivatization of the polyamines may be effected using alkylation agentssuch as those described by EP-A-230893 (Felder), e.g. HalCH₂ COL",HalCH(COOH)CH₂ O Benzyl, or HalCH(COOH)₂ (where Hal is Cl or Br and L"is OH, NHAlkyl or NAlkyl₂ (e.g NHCH₃ or N(CH₃)₂) or HalCH₂ NAlkyl₂ (e.g.ClCH₂ N(CH₃)₂), followed where necessary by deprotection of protectedgroups. Examples of such schemes include ##STR11## Selective alkylationof amines is described by Nordlander et al. Tetr. Lett. (1978) 4987 andJ. Org. Chem. 49: 133 (1984) and by Aspinall et al. JACS 63: (1941).Many other appropriate derivatization procedures are described in theliterature.

For the reductive procedure discussed above, reaction may be of many ofthe same or similar polyamines with aidehyde, carboxyl or carboxylderivative compounds followed by reduction of the amide carbonyl groups,e.g. using sodium cyanoborohydride or diborane, e.g. as in the scheme##STR12##

The resulting thioesters could equally be produced by reaction of anaminocarboxylic acid reagent with a chloroalkylsulphide, e.g. ##STR13##

As mentioned above, the chelants of formula (XIV) can also be producedby amination of polyfunctional reagents. One example of this procedureis given by Huber et al. J. Chem. Soc. Chem. Comm. (1989) 879, i.e.##STR14##

The resulting polyamine can then be converted to a compound of formulaXIV by reaction with HOCH₂ CN followed by hydrolysis. A wide variety ofother polyhalo and amine compounds suitable for use in such reactionsare available commercially or may be prepared using text book methods.

In a similar manner, polyfunctional acids may be reacted withappropriate amines if necessary after activation of the acid groups,reduction of the amide and alkylation will yield chelants of formulaXIV. Commercially available polyfunctional acids utilizable in this wayinclude for example

HOOCBCOOH

where B is --CHOHCH₂ CH₂ --, --(CHOH)₂ --, --(CH₂)₁₋₃ -- or ##STR15##

In order to attach the chelant to a macromolecule, e.g. a protein or acarbohydrate, the chelant may be provided with a reactive side chain(e.g. described by Meares et al. Anal. Biochem. 142: 68(1984), etc).Alternatively attachment can be efected for example using the methodsdeveloped by Salutar Inc. (See for example WO-A-90/12050 and Sieving etal., Bioconjugate Chem. 1: 65-71 (1990)) or the mixed anhydride orcyclic anhydride methods of Krejcarek et al Biochemical and BiophysicalResearch Comm. 77: 881 (1977) or Hnatowich et al. Science 220: 613(1983) etc. Attachment of the chelant may be either directly to themacromolecule or, preferably, to a an intermediate polymer, e.g.poly-L-lysine or polyethylene-imine, onto which a plurality of chelantsmay be loaded, e.g. as discussed in EP-A-331616 (Deutsch).

Thus for example the following macromolecule-linkable chelants aresuggested in the literature: ##STR16##

The tridentate tris-thiols of Holm et al. (see JACS 112: 8015-8023(1990) and JACS 110: 2484-2494 (1988)) also deserve particular mention,especially for the complexation of tetranuclear clusters.

The multinuclear complexes used according to the invention may beprepared by the methods suggested in the literature or by analogousmethods. In particular, novel complexes may be prepared from knowncomplexes by ligand interchange.

Thus, for example for tungsten based multinuclear entities as mentionedabove, oxalatotungstate(V) may be used as a starting material and ligandexchange reactions with calcium chelates of APCAs to precipitate outcalcium oxalate may be carried out. Chromatographic isolation andpurification methods, such as suggested by Ikari (supra) appearparticularly suitable.

The preparation of an intermediate oxalate may be avoided by use ofother literature known methods, e.g. the electrochemical reductionsuggested by Baba et al. Mem. Fac. Tech. Tokyo Metropolitan Univ. 32:3207 (1982).

Other preparative techniques that deserve particular mention include theoxidation of tungstate complexes with the addition of the desiredchelant/complexant as suggested by Chaudhuri (supra) and the reductionof tungstates with reductants and a chelant/complexant (which may haveoxidative or reductive properties) as suggested by Lozano et al. inPolyhedron 31: 25-29 (1984).

Further examples of synthetic routes by which the multinuclear complexesused according to the invention may be prepared include: ##STR17##Molybdenum and tungsten trinuclear aqua complexes [M₃ (μ₃ B)(μ₂ B)₃ (H₂O)₉ ]⁴⁺ (where M is Mo or W and B is O or S) can be prepared by methodsknown from the literature. The co-ordinated waters in these complexescan readily be replaced by chelants xvi to xx to reduce toxicity. Singleor mixed ligand combinations may be used to produce ionic or non-ioniccomplexes. ##STR18## The coordinated water in the tetranuclearaquacomplexes may be substituted by ligands such as chelants i to vii toreduce toxicity. Selected examples are shown below. ##STR19##

Molybdenum and tungsten based tetranuclear aqua complexes (M₄ (μ₃ B)₄(H₂ O)₁₂)^(n+) (where M=W or Mo, B=S or Se and n=4 or 5) can be preparedby various chemical and electrochemical procedures. Tetranucleartungsten complexes may also be prepared by reduction of binuclearcomplexes, e.g. using reductants such as NaBH₄, Na₂ S₂ O₄ and Zn/Hgamalgam and the compound of formula XXIV, by photoirradition of tungstenhexacarbonyl and sodium sulphide in methanol, or of a mixture of atrinuclear complex and tungsten hexacarbonyl in methanol or reaction ofa trinuclear complex and the W(III) aquoion under reducing conditionswith heat or photo-irradition.

For adminstration to human or animal subjects, the multinuclearcomplexes will conveniently be formulated together with pharmaceuticalor veterinary carriers or excipient. The contrast media of the inventionmay conveniently contain pharmaceutical or veterinary formulation aids,for example stabilizers, antioxidants, osmolality adjusting agents,buffers, pH adjusting agents, colorants, flavours, viscosity adjustingagents and the like. They may be in forms suitable for parenteral orenteral administration, for example, injection or infusion oradminstration directly into a body cavity having an external voidanceduct, for example the gastrointestinal tract, the bladder and theuterus. Thus the media of the invention may be in conventionalpharmaceutical adminstration forms such as tablets, coated tablets,capsules, powders, solutions, suspensions, dispersions, syrups,suppositories etc; solutions, suspensions and dispersions inphysiologically acceptable carrier media, e.g. water for injections,will however generally be preferred. Where the medium is formulated forparenteral administration, the carrier medium incorporating themultinuclear complex is preferably isotonic or somewhat hypertonic.Moreover, media for parenteral administration will preferably containsmall quantities, e.g. 0.01 to 10 mole percent relative to themultinuclear complex of free chelants or of weak chelate complexes withphysiologically tolerable chelated species (e.g. Ca²⁺ ); small additionsof sodium or calcium salts may also advantageously be made.

For use as X-ray contrast media, the media of the invention shouldgenerally have a heavy atom content of 1 millimole/l to 5 mole/l,preferably 0.1 to 2 mole/l. Dosages of from 0.5 to 1.5 mmoles/kg willgenerally be sufficient to provide adequate contrast although dosages of0.8 to 1.2 mmoles/kg will normally be preferred.

For scintigraphy, dosages of the radioactive species will generally belower.

Thus in summary the present invention provides a particularly effectivemeans by which contrast media efficiency may be enhanced by increasingthe relative proportion of molecular volume that is occupied by thecontrast enhancing heavy or paramagnetic metal atom. For X-ray contrastmedia in particular, this also enables higher K- edge value atoms thanthe iodine of the now conventional X-ray contrast media to be utilizedeffectively.

The present invention will now be illustrated further by the followingnon-limiting Examples (all ratios and percentages are by weight and alltemperatures are in degrees Celsius unless specified otherwise):

EXAMPLE 1Bis(μ-oxo)(μ-ethylenediaminotetraaceto-N,N')bis(oxotungstate(V)),disodium salt Na₂ [W₂ O₂ (μ₂ O)₂ (μ₂ EDTA)]

Alternative A:

The potassium salt (37 g, 65 mmol) of the oxalato complex of tungsten(V)(prepared according to Collenberg, Z. Anorg. Allg. Chem. 102: 247-276(1918)), sodium acetate (60 g, 441 mmol) and ethylenediaminetetraaceticacid (10 g, 34 mmol) were dissolved in oxygen free water (800 ml) andwarmed to 80°-90° C. under nitrogen. Degassed warm calcium acetatesolution (1M, 150 ml) was added with stirring and the mixture wasallowed to cool. After filtering off the precipitate, degassed bariumacetate solution (1M, 40 ml) was added. A small amount of the immediateprecipitate was filtered off and the filtrate was concentrated in vacuo.The residue was collected on a filter, washed with water and dried. Thismaterial (18.1 g, 21 mmol barium-complex) was dissolved in warm oxygenfree water (2000 ml) and sodium sulfate solution (1M, 25 ml) was added.The mixture was allowed to cool, filtered and concentrated to dryness.The residue was taken up with water (50 ml) and precipitated bysuccessive addition of ethanol.

Yield: 15.17 g (30 %) of the sodium salt of tungsten-EDTA complex.

Purification of 10 g by HPLC afforded the title compounds of Example 1(3 g) and Example 2 (4.25 g)

¹ H-NMR was as reported by Ikari et al., Inorg. Chem. 28: 1248-1254(1989).

Alternative B:

The potassium salt (0.10 g, 0,062 mmol) of the oxalato complex oftungsten(V) (prepared according to Baba et al., Mem. Fac. Tech. TokyoMetropolitan Univ. 32: 3207-3220 (1982)), ethylenediaminetetraaceticacid (0.028 g, 0.124 mmol) and sodium acetate (0.021 g, 0.25 mmol) weredissolved under nitrogen in oxygen free water (1.5 ml) and heated to 100° C. Calcium chloride dihydrate (0.046 g, 0.31 mmol) dissolved in oxygenfree water (2 ml) was added and the mixture allowed to cool. Afterfiltering off the precipitate, barium hydroxide (0.043 g, 0.136 mmol),dissolved in water (2 ml), at pH 4 (acetic acid) was added. The mixturewas concentrated in vacuo to near dryness, the precipitate collected bycentrifugation, washed with two drops of water and dried in vacuo overP₂ O₅.

Yield: 0,044 g (21%) of the barium salt of the complex.

This was dissolved in water (20 ml) with heating, sodium sulfate (0,017g, 0.05 mmol) dissolved in water (2 ml) was added and the precipitateremoved by centrifugation. Concentration of the clear solution todryness gave the title compound quantitatively. HPLC-analysis showed theproduct to be identical with that prepared by alternative A.

EXAMPLE 2(μ-Ethylenediaminotetraaceto-N,N')(μ-oxo)(μ-sulphido)bis(oxotungstate(V)),disodium salt Na₂ [W₂ O₂ (μ₂ O)(μ₂ S)(μ₂ EDTA)]

The potassium salt (37 g, 65 mmol) of the oxalato complex of tungsten(V)(prepared according to Collenberg, Z. Anorg. Allg. Chem. 102: 247-276(1918)), sodium acetate (60 g, 441 mmol) and ethylenediaminetetraaceticacid (10 g, 34 mmol) were dissolved in oxygen free water (800 ml) andwarmed to 80°-90° C. under nitrogen. Degassed warm 1M calcium acetatesolution (150 ml) was added with stirring and the mixture was allowed tocool. After filtering off the precipitate, degassed 1M barium acetatesolution (40 ml) was added. A small amount of immediate precipitate wasfiltered off and the filtrate was concentrated in vacuo. The residue wascollected on a filter, washed with water and dried.

This material (18.1 g, 21 mmol barium - complex) was dissolved in warmoxygen free water (2000 ml) and 1M sodium sulfate solution (25 ml) wasadded. The mixture was allowed to cool, filtered and concentrated todryness. The residue was taken up with water (50 ml) and precipitated bysuccessive addition of ethanol.

Yield 15.17 g (30 %) of the sodium salt of tungsten-EDTA complex.

Purification of 10 g crude material by HPLC afforded 4.25 g of the titlecompound.

¹ H-NMR was as reported by Ikari et al., Inorg. Chem. 28: 447-451(1989).

EXAMPLE 3Bis(μ-sulphido)(μ-ethylenediaminotetraaceto-N,N')bis(oxotungstate(V))disodium salt Na₂ [W₂ O₂ (μ₂ S)₂ (μ₂ EDTA)]

Alternative A

The title compound is prepared and purified according to the procedureof Shibahara et al., 37^(th) Nat. Conf. Coord. Chem., Tokyo, Abstr.1AP06.

Alternative B

1 g of (NH₂)₂ WS₄ (2.87 mmol) was dissolved in 20 ml of H₂ O to give ayellow suspension. 1 g of NaBH₄ on alumina and 10 ml of 6M HCl werealternatively added to the yellow suspension. An immediate dark-brownsuspension was formed, which was then heated at 120° C. under an O₂stream for 15 hours. After cooling the resulting mixture to ambienttemperature, a green solid was removed by filtration. The red-brownfiltrate was treated with 0.6 g of Na₄ EDTA. After the pH of the mixturewas adjusted to 1.2, it was heated at 100° C. for 1 hour. Someprecipitate was observed during this heating period. After removing thesolid by filtration, the yellow-orange filtrate was cooled and left atambient temperature for 4 days, giving orange crystals. The crystalswere washed with a small amount of methanol and then dried in the air.The yield was 0.20 g.

The mass spectral data of this product showed the molecular ions at 791and 753 corresponding to the mass of K[W₂ O₂ S₂ (EDTA)] and [W₂ O₂ S₂(EDTA)]+H, respectively. ¹ H NMR resonances in D₂ O were found at 2.54ppm(s, 4H), 3.14 ppm(d, 2H, J=16.4 Hz), 3.40 ppm(d, 2H, J=17. Hz) and3.52 ppm(d, 4H J=17.1 Hz).

EXAMPLE 4 Bis(μ-oxo)(μ-ethylenediaminotetraaceto-N,N')(oxomolybdenum(V))(oxotungstate(V)), disodiumsalt Na₂ [MoWO₂ (μ₂ O)₂ (μ₂ EDTA)]

The title compound is prepared and purified according to the procedureof Ikari et al. Inorg. Chem. 28: 1248-1254 (1989).

EXAMPLE 5 Preparation of a solution containing the disodium salt ofbis(μ-oxo)(μ-ethylenediaminotetraaceto-N,N')bis(oxotungstate(V))

The salt from Example 1 (2.95 g, 3.85 mmol) was dissolved in water (18ml) and the pH was adjusted to 7 by careful addition of 1M sodiumhydroxide. Water was added to 20 ml, the solution passed through a 0.22μm sterile filter and placed into four 5 ml vials. The solutioncontained 0.20 mmol of the disodium salt ofbis(μ-oxo)(μ-ethylenediaminotetraaceto-N,N')bis(oxotungstate(V)) per ml.The LD₅₀ in mice was found to be 10-14 mmol/kg.

EXAMPLE 6 Preparation of a solution containing the disodium salt of(μ-ethylenediaminotetraaceto-N,N')(μ-oxo)(μ-sulphido)bis(oxotungstate(V))

The salt from Example 2 (2.66 g, 3.40 mmol) was dissolved in water (17ml) and the pH was adjusted to 7.0 by careful addition of 1 M sodiumhydroxide. The solution was passed through a 0.45 μm filter into four 5ml vials.

The solution contained 0.19 mmol of the disodium salt of(μ-ethylenediaminotetraaceto-N,N')(μ-oxo)(μ-sulphido)bis(oxotungstate(V))per ml. The LD₅₀ in mice was found to be approx. 10 mmol/kg.

EXAMPLE 7 Preparation of a solution containing the disodium salt ofbis(μ-sulphido)(μ-ethylenediaminotetraaceto-N,N')bis(oxotungstate(V)

The salt from Example 3 (2.00 g, 2.5 mmol) is dissolved in water (12.5ml) and the pH is adjusted to 7.0 by careful addition of 1M sodiumhydroxide. The solution is filtered into three 5 ml vials.

EXAMPLE 8 Preparation of a solution containing the disodium salt ofbis(μ-oxo)(μ-ethylenediaminotetraaceto-N,N') (oxomolybdenum(V))(oxotungstate(V))

The salt from Example 4 (2.00 g, 2.95 mmol) is dissolved in water (14.7ml) and the pH is adjusted to 7.0 by careful addition of 1M sodiumhydroxide. The solution is filtered into three 5 ml vials.

EXAMPLE 9Bis(μ-oxo)(μ-N,N'-propylenediaminetetraacetato)bis(oxotungstate(V)),barium salt Ba[W₂ O₂ (μ₂ O)₂ (μ₂ EDTA)]

The potassium salt (1.61 g, 1 mmol) of the oxalato complex oftungsten(V) (prepared according to Baba et al., Mem. Fac. Tech. TokyoMetropolitan Univ. 32: 3207-3220 (1982)) and1,2-diaminopropane-N,N,N',N'-tetraacetic acid (0.612 g, 2 mmol) weredissolved under nitrogen in a mixture of 50 ml oxygen-free water andsodium acetate-solution (1M, 8 ml) and heated to 100° C. Calciumacetate-solution (1M, 10 ml) was added with stirring and the mixtureallowed to cool. After filtering off the precipitate, a bariumacetate-solution (1M, 2 ml) was added, the solution was filtered and thetitle compound was precipitated by dropwise addition of ethanol. It wascollected on a filter, washed with 50% aqueous methanol and dried invacuo at 40° C. Yield: 0.413 g (43%) of the pentahydrate.

EXAMPLE 10Bis(μ-oxo)(μ-N,N'-propylenediaminetetraacetato)bis(oxotungstate(V)),sodium salt Na[W₂ O₂ (μ₂ O)₂ (μ₂ EDTA)]

The title compound is prepared by dissolving the barium salt of Example9 in warm water. After addition of a stoichiometric amount of a 1Msodium sulfate solution the mixture is allowed to cool, filtered and thefiltrate concentrated to dryness.

EXAMPLE 11

The title compound was prepared by a slightly modified version of theprocedure described in JACS 108:2757-2758 (1986).

3 g of (NH₄)₂ WS₄ (8.62 mmol) was dissolved in 75 ml of water to give ayellow solution. 3 g of NaBH₄ and 30 ml of concentrated HCl were addedalternatively to the tungsten solution. Upon this addition, an immediatecolor change from yellow to dark brown was observed. The resulting brownsuspension was heated at 100° C. for 2 hours After cooling the mixture,it was filtered to remove a dark brown solid and to obtain a brownfiltrate. The brown solution was loaded on a Sephadex G-15 column, whichresulted in a brown band on top of the column. After a 5-day airoxidation of the brown band, it was eluted with 2M HCl solution. Thesecond purple fraction (λmax=570 nm and 320 nm) was collected andevaporated to dryness under high vacuum at 36°-40° C., which gave darkgrey solid. The product was washed with acetone and dried in the air.The yield was 0.506 g (0.62 mmol, 22%).

The mass spectral data in dithiothrietol(DTT)/dithioerythrietol matrixgives a molecular ion at 1139 equivalent to the mass of [W₃ S₄ (DTT)₃]+2H.

The elemental analysis indicated that the product was W₃ S₄ (H₂ O)₉ Cl₄and contained 2.4% HCl and 3.9% H₂ O.

Calculated: W(52.53%), S(12.22%), Cl(15.86%). Found: W(52.46%),S(12.24%), Cl(15.96%).

EXAMPLE 12 [N(C₂ H₅)₄ ][W₂ S₂ (μ-S)₂ (EDT)₂ ]

This compound was prepared according to a literature procedure. (Inorg.Chem. 23: 4265-4269 (1984)). 0.81 g (2.3 mmol) of (NH₄)₂ WS₄ was addedto 25 ml of N₂ -saturated DMF. The resulting mixture was agreenish-yellow suspension. After adding 0.3 ml (3.6 mmol) of1,2-ethanedithiol (EDT), a bright yellow color formed. The reactionmixture was heated under a N₂ flow at 120° C. in an oil bath for 2hours. After several minutes of heating, the solution become red-orange.At the end of the reaction period, a brownish red suspension was noted,0.63 g of N(C₂ H₅)₄ Cl was then added to the cooled suspension atambient temperature. 20 ml of diethyl ether was added to precipitate theproduct. Brownish red crystals were recovered by filtration and washedwith methanol and then with ether. The addition of more ether (150 ml)to the red-orange filtrate gave more product. All the fractions werethen combined and recrystallized once from methanol. The total yield ofthe product was 0.75 g (1.6 mmol, 69% from (NH₄)₂ [WS₄ ]).

The mass spectral data of this product showed a molecular ion at 681corresponding to W₂ S₄ (EDT)₂ +H.

We claim:
 1. A sterile X-ray contrast medium comprising at least onepharmaceutical carrier or excipient together with a physiologicallytolerable multinuclear tungsten complex of the formula

    (W.sub.3 B.sub.u A.sub.v).sub.x L.sub.w,

wherein W₃ B_(u) A_(v) is a multinuclear entity; each tungsten atom ofwhich is covalently bonded to at least one atom B; each B of which maybe the same or different and is a bridging atom covalently bonded to atleast two tungsten atoms; each A of which may be the same or differentand is a non-bridging atom covalently bonded to a tungsten atom; each Lof which may be the same or different and is a ligand coordinatelybonded to at least one tungsten atom; u is a positive integer of value 2or greater; x and w are positive integers; and v is zero or a positiveinteger, or a physiologically tolerable salt of said tungsten complex.2. A medium as claimed in claim 1 where each A and B is O, S, Se, Te ora protonated or substituted nitrogen or phosphorus atom.
 3. A medium asclaimed in claim 2 wherein the multinuclear entity comprises a unit offormula W₃ (μ₃ B)(μB)₃ wherein each B is O, S, Se, Te or a protonated orsubstituted nitrogen or phosphorus atom.
 4. A medium as claimed in claim3 wherein said chelant is an aminopolycarboxylic acid or an ester oramide thereof or is a macrocyclic polyacylated polyamine.
 5. A medium asclaimed in claim 1 further comprising a free chelant or aphysiologically tolerable salt or a weak complex thereof with aphysiologically tolerable metal ion.
 6. In a method of generatingcontrast-enhanced X-ray images of a subject comprising administering tosaid subject an X-ray attenuating amount of a physiologically tolerablesterile X-ray contrast agent and generating an X-ray image of saidsubject, the improvement comprising administering as said contrast agenta compound having an anion comprising a bridging atom cluster W₃ B_(b)each B is independently an oxygen or sulfur atom, each W is bound to atleast two B atoms, each B atom is bound to at least two W atoms, and bis an integer having a value of at least two.
 7. A method as claimed inclaim 6 wherein said cluster is a W₃ B₄ cluster.
 8. A method as claimedin claim 6 wherein said anion is complexed by a ligand.
 9. A method asclaimed in claim 8 wherein said ligand is an aminopolycarboxylic acid.